Diagnosis of Leishmania infantum infection by Polymerase Chain Reaction in wild mammals

Visceral leishmaniasis is a chronic infectious disease caused by Leishmania infantum (synonym: Leishmania chagasi) and transmitted by the sandfly Lutzomyia longipalpis in Brazil. It is an endemic zoonosis in several regions of the country, including Belo Horizonte (State of Minas Gerais). In urban areas, the domestic dog is susceptible and considered the most important animal reservoir. However, L. infantum has been previously diagnosed in other species, including captive primates and canids. This study aimed to evaluate the presence of the agent DNA in captive animals as well as some free ranging animals from the Zoo-Botanical Foundation of Belo Horizonte by Polymerase Chain Reaction. Eighty one blood samples from primates, carnivores, ruminants, edentates, marsupial, and a monogastric herbivore were analyzed. Three primates Alouatta guariba (brown howler monkey), and two canids Speothos venaticus (bush dog) were positive, demonstrating the importance of leishmaniasis control in endemic areas for preservation of wildlife species in captivity.

Molecular survey of porcine teschovirus, porcine sapelovirus, and enterovirus G in captive wild boars (Sus scrofa scrofa) of Paraná state, Brazil

AbstractPorcine teschovirus (PTV), porcine sapelovirus (PSV), and enterovirus G (EV-G) are infectious agents specific to pig host species that are endemically spread worldwide. This study aimed to investigate the natural infection by these porcine enteric picornaviruses in wild boars (Sus scrofa scrofa) of Paraná state, Brazil, and to evaluate peccaries (Pecari tajacu and Tayassu pecari) as alternative host species for these viruses. Fecal samples (n=36) from asymptomatic wild boars (n=22) with ages ranging from 2 to 7 months old (young, n=14) and 2 to 4 years old (adult, n=8) and from peccaries (6 to 8 months old, n=14) were collected from a farm and a zoo, respectively, both located in Paraná state. Reverse transcription-polymerase chain reaction (RT-PCR) and nested-PCR (n-PCR) assays targeting the 5'non-translated region of the virus genome were used for screening the viruses. Porcine enteric picornaviruses were detected in 12 out of the 22 wild boar fecal samples. According to each of the viruses, EV-G was most frequently (11/22, 50%) detected, followed by PTV (10/22, 45.5%) and PSV (4/22, 18.2%). Regarding the age groups, young wild boars were more frequently (9/14, 64.3%) infected with PTV, PSV, and EV-G than adult animals (3/8, 37.4%). One n-PCR amplified product for each of the viruses was submitted to sequencing analysis and the nucleotide sequences were compared with the related viruses, which showed similarities varying from 97.7% to 100% for PTV, 92.4% to 96.2% for PSV, and 87.1% to 100% for EV-G. Peccaries tested negative for the viruses and in this study they did not represent infection reservoirs. This study is the first to report the molecular detection of PTV, PSV, and EV-G from captive wild boars in a South American country and the first to screen peccaries as alternative host species for porcine enteric picornavirus.

Outbreaks of canid herpesvirus 1 disease in puppies in southern Brazil

Abstract: Canid herpesvirus 1 (CHV-1) is a widespread pathogen of dogs and produces infertility, abortions and severe systemic disease in young puppies. Clinical data indicate the circulation of CHV-1 among Brazilian dogs yet definitive diagnosis has rarely been accomplished. This article describes the clinicopathological findings of four independent cases/outbreaks of neonatal disease by CHV-1 in Bulldog puppies followed by virus identification and genetic characterization. Three events occurred in a kennel holding dogs of different breeds at reproductive age (March 2013, October 2013 and April 2014). Puppies from three French or English Bulldog litters, aging 9 to 30 days were affected, presenting dyspnea, agonic breathing, pale mucous, abdominal pain and tension, evolving to death within about 24 hours. At necropsy, the puppies presented necrohemorrhagic hepatitis, multifocal and moderate necrohemorrhagic nephritis and fibrinonecrotic interstitial pneumonia. Virus isolation was positive in clinical specimens from one litter and CHV-1 DNA was detected by PCR in tissues from all four cases. Virus-neutralizing assays with samples of the affected kennel revealed 9/12 adult animals with high antibody titers to CHV-1. Nucleotide sequencing of glycoprotein B, C and D genes revealed 99-100% of identity among the viruses and with CHV-1 sequences available in GenBank. Phylogenetic analyses of gC sequences showed a segregation of the samples, even among three isolates from the same kennel. These findings support CHV-1 infection as the cause of disease and death in these dog litters, reinforcing the need for correct etiologic diagnosis, prevention and immunization against CHV-1 in dogs from Southern Brazil.

Occurrence of antibodies against Leptospira spp. In free-ranging wild canids from the Brazilian savanna

Abstract: The Brazilian savanna, also known as Cerrado, is one of the world's richest and most ecologically invaluable tropical savanna regions. There are few studies in Brazil about the diseases that affect the wild canids of this biome, which may be harmful to wildlife populations and public health. The aim of this study was to evaluate the occurrence of antibodies against Leptospira spp. in three Cerrado wild canids species using the microscopic agglutination test (MAT). Serum samples were tested from 19 crab-eating foxes (Cerdocyon thous), 14 maned wolves (Chrysocyon brachyurus), and seven hoary foxes (Lycalopex vetulus), all free-ranging animals found in the municipalities of Araguari and Uberlândia, Minas Gerais State, and Cumari, Goiás State, Brazil. Fourteen (35%) of these samples were seropositive. The most frequent serovars detected in the samples were Copenhageni and Hardjo, but reactions to the serovars Autumnalis, Grippotyphosa, Hebdomadis, Wolffi, and Icterohaemorrhagiae also occurred. Notwithstanding other reported results, this study is the first to report the presence of antibodies against Leptospira spp. in L. vetulus. The three species of wild canids examined may act as potential hosts for several serovars of leptospira in Brazil's savanna environment.

Germination ecology of wild onion: a rainfed crop weed

Asphodelus tenuifolius is becoming a more common weed in rain-fed area in Pakistan. Laboratory and greenhouse experiments were conducted to determine the effect of different environmental factors on germination and emergence of A.tenuifolius. Results showed that A.tenuifolius can tolerate a wide range of varying environmental factors. Greatest percentage of germination (80%) was recorded at 15 ºC constant temperature; however, considerable germination occurred at 20 and 25 ºC. Light for 10 h photoperiod stimulate germination of Asphodelus tenuifolius compared with complete darkness. Germination was totally inhibited at osmotic stress higher than -0.8 MPa. There was no significant difference in germination at pH 6 to 8; however, there was a slightly decrease at pH 9, compared with distilled water. Asphodelus tenuifolius was very sensitive to salinity; however, a few seeds of A.tenuifolius were able to germinate even at 150 mM NaCl concentration. Greatest emergence occurred with seed placed at soil surface and emergence decreased with increase in burial depth. No emergence occurred from 4 cm or greater. This information may aid in developing tools and strategies for management.

Wild onion (Asphodelus tenuifolius) competition in rainfed chickpea-chickpea cropping system

Chickpea yield potential is limited by weed competition in typical chickpea growing areas of Pakistan where zero tillage crop grown on moisture conserved from rains received during the months of September and August. The objective of this work was to evaluate the growth and yield characteristics of chickpea grown in coexistence with increasing densities of wild onion (Asphodelus tenuifolius). The experiment was comprised of six density levels viz. zero, 20, 40, 80, 160 and 320 plants m-2 of A. tenuifolius. A decrease in chickpea primary and secondary branches per plant, pods per plant, seeds per pod, 100-seed weight and seed yield was observed due to more accumulation of dry matter per increasing densities of A. tenuifolius. The increase in A. tenuifolius density accelerated chickpea yield losses and reached the maximum values of 28, 35, 42, 50, 58 and 96% at 20, 40, 80, 160 and 320 A. tenuifolius plants m-2, respectively. The yield loss estimation model showed that chickpea losses with infinite A. tenuifolius density were 60%. Yield reduction could be predicted by 2.52% with increase of one A. tenuifolius plant m-2. It is concluded that A. tenuifolius has a strong influence on chickpea seed yield and showed a linear response at the range of densities studied.

Meiotic behavior in wild and domesticated species of Passiflora

The meiotic behavior of fourteen Passiflora taxa was analyzed. The species were grouped according to the n value (6, 9 and 12) for statistical studies. Some species presented tetravalent associations or univalent chromosomes in diakinesis, bivalent formation prevailing. The qui-square test revealed significant differences in the chiasma frequency among species for n = 9 and n = 6 groups. There was predominance of interstitial chiasmata in almost all studied species. The n = 12 group was the only one whose meiotic behavior was considered similar due to the quantity of chiasmata per cell, tendency of interstitial chiasma localization. Some species presented meiotic irregularities, such as laggard and precocious chromosomes in meiosis I. In telophase II the percentages of meiotic irregularities was low. Irregularities in the spindle orientation were presented in higher percentages in the end of meiosis II, and were also responsible for post-meiotic abnormal products. The irregularities observed during meiosis can have influence on the percentage of sterile pollen grains and success of interspecific crossings in Passiflora species.

Hybridization among wild passionflower species

(Hybridization among wild passionflower species). Passion fruits are appreciated for their ornamental value, since their flowers are showy and display a wide variety of colors. In addition, many hybrids have been produced and used in other countries. The genotypes used in selection of plants with ornamental characteristics are hybrid progenies which are used in various crossing strategies. Thus, the aim of this work was to obtain interspecific hybrids, perform backcrossing and obtain progenies from crossings between hybrids, and to determine the reproductive compatibility between the progenitors involved. The percentage of fertilized flowers, germination, and the number of fruits, seeds and plants obtained through crossing were recorded. A series of 374 crossings involved seven species and two hybrids. Crossings such as Passiflora gibertii N. E. Brown vs. P. kermesina Link & Otto and P. gibertii vs. P. alata Curtis did not produce seeds. The largest percentage of fertilized flowers (86%) was recorded for the crossing P. gardneri Mast.vs. P. cincinnata Mast.; yet, the seeds produced did not show endosperm. Interspecific hybrids were obtained from the crossings P. gardneri vs. P. alata, P. watsoniana Mast.vs. P. alata, P. watsoniana vs. P. gardneri and P. gardneri vs. P. gibertii. Seeds generated from backcrossings involving the hybrids P. sublanceolata (sin. P. palmeri var. sublanceolata (Killip) J. M. MacDougal) vs. P. foetida var. foetida L. (HD13-133 and HD13-141) and F2 reached high germination percentages.

Overcoming crossing barriers between cassava, Manihot esculenta Crantz and a wild relative, M. pohlii Warwa

The use of mentor pollen has enabled successful hybridization between cassava, Manihot esculenta Crantz, and the wild species M. pohlii Warwa. Killed pollen of a cross compatible type produced by freeze-thawing was mixed with incompatible pollen and the mixes were dusted on stigmas. This treatment resulted in production of seed in 4.9% of the total pollinations, compared to 0% in the case of untreated pollinations. The use of a bridge species, M. neusana Nassar, through the hybrid M. pohlii and M. neusana also proved successful in overcoming interspecific barriers between cassava and M. pohlii.

Morphometry and acetylcholinesterase activity of the myenteric plexus of the wild mouse Calomys callosus

The myenteric plexus of the digestive tract of the wild mouse Calomys callosus was examined using a histochemical method that selectively stains nerve cells, and the acetylcholinesterase (AChE) histochemical technique in whole-mount preparations. Neuronal density was 1,500 ± 116 neurons/cm2 (mean ± SEM) in the esophagus, 8,900 ± 1,518 in the stomach, 9,000 ± 711 in the jejunum and 13,100 ± 2,089 in the colon. The difference in neuronal density between the esophagus and other regions was statistically significant. The neuron profile area ranged from 45 to 1,100 µm2. The difference in nerve cell size between the jejunum and other regions was statistically significant. AChE-positive nerve fibers were distributed within the myenteric plexus which is formed by a primary meshwork of large nerve bundles and a secondary meshwork of finer nerve bundles. Most of the nerve cells displayed AChE activity in the cytoplasm of different reaction intensities. These results are important in order to understand the changes occurring in the myenteric plexus in experimental Chagas' disease

Plasma cortisol levels in captive wild felines after chemical restraint

Eight Panthera onca (Po), 13 Felis concolor (Fc), 7 Felis yagouaroundi (Fy), 7 Felis tigrina (Ft) and 5 Felis pardalis (Fp) specimens from São Paulo State zoos were used. All animals were restrained with darts containing 10 mg/kg ketamine and 1 mg/kg xylazine. Venous blood samples were collected as soon as possible (within 15-20 min) and serum was frozen until the time for cortisol quantification. Cortisol was determined using a solid phase radioimmunoassay with an intra-assay coefficient of 8.51%. Data were analyzed statistically by the Kruskal-Wallis test, followed by Dunn's multiple comparisons test, and the one-sample t-test, with the level of significance set at P<0.05. Data are reported as means ± SEM. Cortisol levels differed among the captive felines: Po = 166 ± 33a, Fc = 670 ± 118b, Fy = 480 ± 83b, Ft = 237 ± 42ab, Fp = 97 ± 12a nmol/l (values followed by different superscript letters were significantly different (P<0.001)). Since most of the veterinary procedures on these species involve chemical restraint, these results show the necessity of preventive measures in order to minimize the effect of restraint stress on more susceptible species

Studies on the intrinsic nervous system of the wild rodent Calomys callosus digestive tract. II: The submucous plexus

The submucous plexus of the normal small and large intestine of Calomys callosus was studied by NADH and AChE histochemical techniques and by transmission and scanning electron microscopy. The plexus contains (mean ± SD) 7,488 ± 293 neurons/cm2 in the duodenum, 5,611 ± 836 in the jejunum, 2,741 ± 360 in the ileum, 3,067 ± 179 in the cecum, and 3,817 ± 256 in the proximal colon. No ganglia or nerve cell bodies were seen in the esophagus, stomach, distal colon or rectum. The neurons are pear-shaped with a round or oval nucleus and the neuronal cell profile areas were larger in the large intestine than in the small intestine. Most of the neurons display intense AChE activity in the cytoplasm. AChE-positive nerve fibers are present in a primary meshwork of large nerve bundles and in a secondary meshwork of finer nerve bundles. At the ultrastructural level, the ganglia are irregular in shape and covered with fibroblast-like cells. The nucleoplasm of the neurons is finely granular with a few condensations of chromatin attached to the nuclear envelope. In the neuropil numerous varicosities filled with vesicles of different size and electron densities are seen. The pre- and post-synaptic membrane thickenings are asymmetric. Characteristic glial cells with oval nuclei and few organelles are numerous. These data provide a detailed description of this submucosal meshwork.

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

Group B rotaviruses (RV-B) were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8) were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

Phenolic compounds and antioxidant activity of hydroalcoholic extracts of wild and cultivated murtilla (Ugni molinae Turcz.)

Over the last decade a considerable increase in the number of studies addressing the use of antioxidants from natural sources has led to the identification and understanding of the potential mechanisms of biologically active components. This results from the fact that they can be used to replace synthetic antioxidants commonly used in food. Murtilla (Ugni molinae Turcz) is a native berry grown in Chile, and in the present study, the phenolic composition and antioxidant activity of its fruits were studied. Hydroalcoholic extracts of dehydrated fruits from two genotypes of murtilla (Ugni molinae Turcz.) were produced. Extracts of wild murtilla and 14-4 genotype fruits had 19.35 and 40.28mg GAE/g for Total Phenolic Compounds, 76.48, and 134.35μmol TEAC/g for DPPH, and 157.04 and 293.99 μmol TEAC/g for ABTS, respectively. Components such as quercetin, epicatechin, and gallic, benzoic and hydrocaffeic acids were identified by CG/MS analysis. All of them showed antioxidant activity. Therefore, it is possible to say that the hydroalcoholic extracts of murtilla have antioxidant potential to be used in lipidic food.